Iron is required for nearly all forms of life, however due to its toxic and reactive properties, it must be transported throughout the body chelated by other molecules to prevent the generation of reactive oxygen species and cause extensive cellular damage. Despite the fact that a number of proteins have been identified that are involved in iron transport and homeostasis, the mechanisms by which they are regulated communicate with each other remain largely unknown. Utilizing small molecules identified in chemical genetics screens to inhibit iron transport has provided initial insight into these mechanisms. The focus of this proposal is to further characterize the mechanism of action of one specific compound, DB38. DB38 inhibits iron transport in mammalian cell culture by promoting degradation of the transferrin receptor and also inhibiting the ferrous iron transporter, divalent metal transporter-1 (DMT1). We have also shown that internalization and degradation of TFR1 by DB38 are sensitive to depletion of membrane cholesterol suggesting that DB38 targets TFR1 to lipid raft microdomains. By dissecting this pathway, we will broaden the scope of membrane biology by defining a unique pathway of TFR1 endocytosis in the presence of DB38. To discover novel proteins sensitive to DB38 treatment we will utilize a genome-wide shRNA screen combined with our fluorescence based inhibition of iron transport assay. Together, we will further our understanding of the mechanism and regulation of several (and currently unknown) proteins involved in iron uptake, transport, and sensing.
Ph.D., 2009, University of Vermont, Burlington, VT